II.A.1. Collection, Screening, and Characterization of Microalgae by
Experiments were also con d ucted in an attempt to identify the chemical components of SERI Type I and Type II media most important for controlling the growth of the various algal strains. Bicarbonate and divalent cation concentrations were found to be important determinants in controlling the growth of Boekelovia sp. (BOEKE1) and Monoraphidium (MONOR2). The growth rate of MONOR2 increased by more than five-fold as the bicarbonate concentration of Type II/25 medium was increased from 2 to 30 mM, and the growth of BOEKE1 by approximately 60% over this range. These results make sense, since media enriched in bicarbonate would have more dissolved carbon available for photosynthesis. An unexpected finding was that there was a decrease of nearly 50% in the growth rate of BOEKE1 as the divalent cation concentration increased from 5 mM to 95 mM (in Type I/10 medium containing aifered^rnounts of calcium and magnesium). The effects of magnesium and calcium
Thecei®ife*tontsth£ growth oratoOiwerg dwieemisd poonoulnuees inrheponenufts gí(^iwalEteP:hasera;ítdhfi?fgctfleufh§Stha tsweers for impteti&M a7 dayduftiSW isneeto foe 2ypeof wnagengvall afbierfon triief^Qi;Ullt3yaltigintOvflnti©(ifiepOeti0l tih e lipid content of the cells, but this was not always the case. The highest lipid content occurred with NAVIC1, which increased from 22% in exponential phase cells to 49% in Si-deficient cells and to 58% in N-deficient cells. For the green alga MONOR2, the lipid content increased from 22% in exponentially growing cells to 52% for cells that had been N-starved for 7 days. CHAET14 also exhibited a large increase in lipid content in response to Si and N deficiency, increasing from 19% to 39% and 38%, respectively. A more modest increase occurred for nutrient-deficient AMPHO1 cells, whereas the lipid content of CYCLO2 was similar in exponential phase and nutrient-deficient cells, and actually decreased in AMPHO2 as a result of nutrient deficiency.
These results suggested that high lipid content was indeed achievable in many strains by manipulating the nutrient levels in the growth media. However, these experiments did not provide information on actual lipid productivity in the cultures, which is the more important factor for developing a commercially viable biodiesel production process. This lack of lipid productivity data also occurred with most of the
ASP subcontractors involved in strain screening and characterization, but was
Pubundtiohsi:ndable because the process for maximizing lipid yields from microalgae grown in mass culture never was optimized. Therefore, there was no basis for Barclay, B.; Nagle, N.; Terry, K. (1986) "Screening microalgae for biomass production designing experiments to estimate lipid productivity potential potential: protocol modification and evaluation." FY 1986 Aquatic Species Program
Annual Report, Solar Energy Research Institute, Golden, Colorado, BERte/ayW313071,pp^ N40e, NJ Weissman, J.C.; Goebel, R.P. (1987) "Potential of new strains of marine and inland saline-adapted microalgae for aquaculture." J.
World. Aquaculture Soc. 18:218-228.
II.A.1.e. Collection and Screening Activities - 1986 and 1987
SERI in-house algal strain collection and screening efforts during 1986-1987 were focused in three separate areas. First, detailed characterization of previously collected strains continued. Second, because the desert southwest sites targeted for biodiesel production facilities can be quite cool during the winter, a new effort to collect strains from cold-water sites was initiated. Finally, a strategy was developed and implemented to reduce the number of strains that had accumulated as a result of Sírsi:uhathctstibs0htfacted research efforts, which allowed researchers to focus on
ElfffigddftMl5 snfe^s^coflected previously from warm-water sites that grew well during the initial screening procedures were characterized with respect to temperature and salinity tolerances, growth rates, and lipid content under various conditions. These strains were Chaetoceros muelleri (strains CHAET6, CHAET9, CHAET10, CHAET15, and CHAET39), Cyclotella cryptica (CYCLO4), Pleurochrysis carterae (PLEUR1), and Thalassiosira weissflogii (THALA2). Each strain was grown in a variety of temperature-salinity combinations by the use of a temperature-salinity gradient table. The maximal growth rate achieved under these conditions occurred
with CHAET9, which exhibited a growth rate of 4.0 doublings- day . The remaining
strains all had maximum growth rates that exceeded 1.4 doublings- day , and several grew at rates exceeding 2.5 doublings- day (i.e., CHAET6, CHAET10, and CHAET39). All had an optimal temperature of 30° C or higher, except for PLEUR1 and THALA2, which had optimal temperatures of 25° C and 28°C, respectively. Most of the strains grew well in a wide range of salinities (e.g., five of the eight strains exhibited a
growth rate greater than one doubling • day at
conductivities between 10 and 70 mmho • cm ). With respect to the effect of water type on growth, CHAET39, CYCLO4, and PLEUR1 grew best on SERI Type I medium. On the other hand, CHAET6, CHAET9, and CHAET10 grew best in SERI Type II medium, but also exhibited good growth on Type I medium and artificial seawater. CHAET15 and THALA2 achieved maximal growth rates on artificial seawater, and,
Thm with PoLEURs stwiW ^syranTywere ^es(0iumg.terTn?igaeedreaoi-l tsexaponne mMy highlight the need to have a variety of algal strains available for the specific water growing cells, as well as for cells that were grown under nutrient-limited conditions.
resources that would be available for mass culture in various locations.
Nitrogen deficiency led to an increase in the lipid contents or CHAET6, CHAET9,
CHAET 10, CHAET15, CHAET39, and PLEUR1. The mean lipid content of these strains increased from 11.2% (of the total organic mass) in nutrient-sufficient cells to
22.7% after 4 days of N deficiency. Silicon deficiency led to an increase in the lipid content of all strains (although in some cases the increase was small and probably not statistically significant). The mean lipid content of the eight strains increased s. cells to 23.4% in Si-deficient cells. A few strains
More moefolipgâi pestas ftsc aCHAETu CMPtheandsPfeEUR1 ■ twhichpdbd fooe
çfodf cmserâ ohaaMe i^euved^xrta®egfowtiatu?ra(tiifi o xp'erience high temperatures; indeed, one subcontractor, Keith Cooksey (Montana State University) specifically searched for thermophilic strains isolated from hot springs. This was because the temperatures of production ponds in the southwestern United States during the prime growing season were expected to reach high levels; thus the production strains would have to thrive under such conditions. However, temperatures in this region
C0lBdqnBe^t}y,walgreSl0gS waU tahi tane de asyeSE Wasglaln?glt0n5 rando to 0tlBlget;oactagll|go;ndl Waaeastelizg)utr0ilís fraimoenld-iW8ee Jhab^;|ctl^s were enriched with N, P, trace metals, and vitamins; artificial media were not used in the initial selection protocol for these experiments. The rotary screening apparatus was maintained at 15° C for the duration of the screening process by including a copper cooling coil inside the screening chamber. The cultures were incubated for 5-10 days, which is longer than for warm-water strains because of the slower growth at the cooler temperature. This procedure created a problem, however, in that many more strains survived the selection process than when 30° C was used as the selection temperature. As a consequence, separating strains from each other and identifying which were the best for further characterization were more difficult.
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